Developing and producing biopharmaceuticals typically involve several purification steps where cell debris, broken particles, other contaminants and clusters etc. should be removed so that the final product contains only the desirable primary particles. The purity and dispersion of the primary particles of interest (i.e. non-clustered primary particles) in the final product is important for its quality and efficacy. To quantitatively assess the purity is hence of importance for the final product but also during the upstream development and production processes to evaluate the efficacy and effect of each purification step. Electron microscopy is a method by which sub-visible particles can be imaged at a resolution sufficient to identifying the particles of interest (primary particles) as well as undesirable debris, contaminants and clusters in the sample. An objective quantitative measure of how pure a sample of sub-visible particles such as virus particles, virus-like particles, inorganic beads and other nanoparticles and micro-particles from liquid samples is important in many processes. For example, modified virus vectors are commonly used in gene therapy applications and modified virus particles are used as vaccines. However, the currently available methods for quantitatively assessing the purity are not very accurate and often involve manual steps that may distort the final result. There is a need for a more effective and reliable method to assess and measure the purity of liquid samples that contain sub-visible primary particles and contaminants/debris.
The method of the present invention provides a solution to the above-outlined problems. More particularly, the method is for quantification of purity of sub-visible particle samples. A sample to be analyzed is placed in an electron microscope to obtain an electron microscopy image of the sample. The sample contains objects of primary particles as well as debris. Debris could be broken or parts (sub-units) of primary particles, and/or contaminants, and/or primary particle or debris clusters or aggregates, and or left-over material from the production phase. The objects in the image are enhanced and have sizes that are different from a size range of primary particles and sizes that are within the size range of primary particles. The objects in the image are detected as being primary particles or debris. The detected primary particles are excluded from the remaining objects so that the objects detected as debris contain only debris and no primary particles. A first total area (T1) of the detected debris is measured. A second total area (T2) of the detected primary particles is measured. A ratio of the first total area (T1) to the second total area (T2) is calculated to determine a quantitative measurement of purity of the sample.
In another embodiment, the edges of objects in the image are enhanced and the objects have a size that is substantially similar to a size range of primary particles. A roundness of the objects is analyzed to identify primary particles.
In another embodiment, objects in the image that have a shape that is substantially similar to that of primary particles are identified as primary particles.
In another embodiment, the edges of objects in the image are enhanced and the objects have a size that is substantially similar to a size range of primary particles and a radial density profile of the objects is analyzed to identify primary particles.
In yet another embodiment, the edges of objects in the image are enhanced and the objects have a size that is substantially similar to a size range of primary particles, and a signal-to-noise ratio at the border of the objects are analyzed by measuring an average intensity of an interior of the objects compared to an average intensity just outside the objects.
In another embodiment, the edges of objects in the image are enhanced and the objects have a size that is substantially similar to a size range of primary particles and a local contrast of the objects are measured by analyzing a sharpness of an outer edge of the objects.
In another embodiment, the edges of objects in the image are enhanced and the objects have a size that is substantially similar to a size range of primary particles and the structure of the objects is measured by means of texture analysis to identify primary particles.
In another embodiment, the structure of the objects in the image is measured by means of texture analysis and analyzed to identify primary particles.
In another embodiment, a sample that contains virus particles or virus-like particles is placed in the electron microscope.
In yet another embodiment, the image is filtered with two smoothing filters to create a first filtered image and a second filtered image and subtracting the first filtered image from the second filtered image.